Project Summary Resistance to therapy is the major clinical challenge in pancreatic cancer therapy. Low efficiency in drug delivery and aggressive tumor biology are the major causes of a poor therapeutic response. It is well known that the presence of a dense fibrous tumor stroma creates a drug delivery barrier and promotes aggressive biology and drug resistance in pancreatic cancer cells. The objective of our translational research project is to develop a new generation of receptor-targeted and protease active targeting ligand and stroma-breaking nanoparticle drug delivery platform for effective treatment of advanced pancreatic cancer. Our extensive research led to the development of a new tumor targeting approach using a recombinant uPAR targeting ligand containing the amino terminal fragment (ATF) of uPA fused with the catalytic domain of matrix metalloproteinase- 14 (ATFmmp14). Novel designs of ATFmmp14 ligand not only ensure enhanced intratumoral delivery of therapeutic agents, but also enable breaking tumor stroma cellular and extracellular matrix barriers. We also developed a biodegradable hyaluronic acid nanoparticle carrying an active metabolite of irinotecan, SN38 (HANP/SN38). Our results demonstrated that ATFmmp14 conjugated HANP/SN38 significantly enhanced drug delivery to tumors in KRAS-driven transgenic mouse and human pancreatic PDX tumor models. Importantly, the nanoparticle-drug migrated out of tumor stroma and penetrated through the basement membrane lining ductal cancer cells to enter tumor cells. As a result, systemic delivery of ATFmmp14-HANP/SN38 significantly inhibited tumor growth. In comparison with current combination therapies (FOLFRINOX or gemcitabine-Nab-Paclitaxel), ATFmmp14-HANP/SN38 treatment significantly prolonged survival in the PDX tumor models derived from drug resistant pancreatic cancer patients. To translate this novel approach for target therapy in cancer patients, in this phase 1 study, we will develop a new generation of tagless human ATFmmp14 ligands that are optimized for the GMP production and have high binding affinity, target specificity and MMP14 enzymatic activity. The proposed studies in Aim 1 will engineer a stable ATFmmp14(R2) expressing CHO cell line and a B. subtilis secretable protein expression system for large scale production of tagless ATFmmp14(R2). We will then evaluate and compare their production procedure, protein yield, target specificity, and MMP14 activity in vitro and in vivo to select the most suitable targeting ligand for drug development (Aim 2). Finally, therapeutic effect of ATFmmp14(R2) will be determined in a pancreatic PDX tumor model (Aim 3). The milestones of this phase I research project are: 1) to develop ATFmmp14-HANP(R2) as a stroma-breaking and targeted nanoparticle drug delivery platform for future commercialization; and 2) to develop a targeted nanoparticle drug, ATFmmp14(R2)- HANP/SN38, for the Phase II study aimed at the Investigational New Drug (IND)-enabling preclinical studies for the translational development of ATFmmp14-HANP/SN38 for targeted therapy of advanced pancreatic cancer.